The present invention relates to a method of predicting the evolutionary potential of a mutant resistance gene, the resulting mutant resistant genes and their expression products, as well as a method of screening a candidate drug for activity against a pathogen including a mutant resistance gene and a method of assessing the potential longevity of a candidate drug.
Antibiotics have proven to be one of medicine""s most effective tools in combating disease, but their utility is constantly being challenged by the emergence of antibiotic-resistant target organisms and their future effectiveness is now in doubt. Because of their efficiency, specificity, and general absence of toxicity, xcex2-lactam antibiotics account for about 50% of global antibiotic consumption (Livermore, xe2x80x9cAre all beta-lactams Created Equal?xe2x80x9d Scand. J. Infecl. Dis. SuDDI. 101: 33-43 (1996); Matagne et al., xe2x80x9cCatalytic Properties of Class A beta-lactamases: Efficiency and Diversity,xe2x80x9d Biochem. J. 330:581-598 (1998)). Since the clinical introduction of benzylpenicillin about 50 years ago the efficiency of xcex2-lactams has been continuously challenged by the emergence of resistant pathogens. As a result new molecules have been progressively introduced with modifications that are increasingly different from the original penicillin (Matagne et al., xe2x80x9cCatalytic Properties of Class A beta-lactamases: Efficiency and Diversity,xe2x80x9d Biochem. J. 330:581-598 (1998)). Genes for resistance to xcex2-lactams are typically plasmid-borne and encode enzymes, called xcex2-lactamases, that degrade and inactivate xcex2-lactam antibiotics. Among plasmid-borne resistance genes the TEM-1 xcex2-lactamase is the most prevalent, accounting for 75% of the xcex2-lactamase in Gram negative organisms worldwide (Amyes, xe2x80x9cGenes and Spectrum: The Theoretical Limits,xe2x80x9d Clin. Infect. Dis. 27 Suppl 1:S21-28 (1998)). The success of TEM-1 xcex2-lactamase and its relatives SHV-1, TEM-2 and OXA-1 is partly the result of its location on plasmids with insertion elements that permitted very rapid dissemination of the xcex2-lactamase genes, and partly the result of continued evolution of the enzyme itself in response to the introduction of new drugs, particularly the cephalosporins and extended-spectrum xcex2-lactams known as third generation cephalosporins.
Another group of xcex2-lactamases, the Class C xcex2-lactamases, are generally quite active toward cephalosporins, including the third generation derivatives, but have not been taken as a serious threat until recently, because the genes for Class C xcex2-lactamases are typically located on chromosomes rather than on plasmids. The chromosomal ampC genes are found in a variety of Gram negative bacteria, including both the Enterobacteriaceae and Pseudomonas species. The ampC genes are typically expressed at a low level, as in E. coli, or are inducible by penicillins and early generation cephalosporins but not inducible by third and fourth generation cephalosporins. Over the last decade, however, it has been found that Gram negative pathogens that are hyper-producers of ampC xcex2-lactamases are resistant to all but a few of the most recently introduced xcex2-lactam antibiotics (Livermore, xe2x80x9cAre all beta-lactams Created Equal?xe2x80x9d Scand. J. Infect. Dis. Suppl. 101: 33-43 (1996)). By now 25-50% of Enterobacter isolates from ICU patients in many major Western and Far Eastern hospitals are AmpC hyper-producers and are resistant to all penicillins and cephalosporins except imipenem, meropenem, and temicillin (Livermore, xe2x80x9cAre all beta-lactams Created Equal?xe2x80x9d Scand. J. Infect. Dis. Suppl. 101: 33-43 (1996)). Even more worrying are several recent reports of derepressed ampC genes located on plasmids found in pathogenic Gram negative bacteria (Bauernfeind et al., xe2x80x9cCharacterization of the Plasmidic beta-lactamase CMY-2, which is Responsible for Cepharnycin Resistance,xe2x80x9d Antimicrob. Agents Chemother. 40:221-224 (1996); Horii et al., xe2x80x9cCharacterization of a Plasmid-borne and Constitutively Expressed blaMOX-1 Gene Encoding AmpC-type beta-lactamase,xe2x80x9d Gene 139:93-98 (1994); Jacoby et al., xe2x80x9cMore Extended-spectrum beta-lactamases,xe2x80x9d Antimicrob. Agents Chemother. 35:1697-1704 (1991); Papanicolaou et al., xe2x80x9cNovel Plasmid-mediated beta-lactamase (MIR-1) Conferring Resistance to Oxyimino- and alpha-methoxy beta-lactams in Clinical Isolates of Klebsiella pneumoniae,xe2x80x9d Antimicrob. Agents Chemother. 34:2200-2209 (1990)). Although Aymes (xe2x80x9cGenes and Spectrum: The Theoretical Limits,xe2x80x9d Clin. Infect. Dis. 27 Suppl 1:S21-28 (1998)) suggests that AmpC xcex2-lactamases at present do not seem efficient enough to cause widespread clinical problems, others (Lindberg et al., xe2x80x9cContribution of Chromosomal beta-lactamases to beta-lactam Resistance in Enterobacteria,xe2x80x9d Rev. Infect. Dis. 8 Suppl 3:S292-304 (1986); Morosini et al., xe2x80x9cAn Extended-spectrum AmpC-type beta-lactamase Obtained by in vitro Antibiotic Selection,xe2x80x9d FEMS Microbiol. Lett. 165:85-90 (1998); Pitout et al., xe2x80x9cAntimicrobial Resistance with Focus on beta-lactam Resistance in Gram-negative Bacilli,xe2x80x9d Am. J. Med. 103:51-59 (1997)) are quite concerned that the AmpC xcex2-lactamases constitute a pool from which clinically significant resistant strains may well emerge. That concern is exacerbated by the finding that in a clinical isolate of Enterobacter cloacae the AmpC xcex2-lactamase has extended its substrate range to include the oxyimino xcex2-lactams as the result of a tandem duplication of three amino acids (Nukaga et al., xe2x80x9cMolecular Evolution of a Class C beta-lactamase Extending its Substrate Specificity,xe2x80x9d J. Biol. Chem. 270:5729-5735 (1995)). More recently, Morosini and his colleagues (Morosini et al., xe2x80x9cAn Extended-spectrum AmpC-type beta-lactamase Obtained by in vitro Antibiotic Selection,xe2x80x9d FEMS Microbiol. Lett. 165:85-90 (1998)) applied direct selection to isolate a mutant of an Enterobacter cloacae AmpC xcex2-lactamase in which the activity toward a fourth generation cephalosporin had increased 267 fold as the result of a single amino acid replacement. Together, the fact that mutations to hyper-expression of AmpC occur readily and the facts that hyper-expressed ampC xcex2-lactamases already confer resistance to penicillins and third generation cephalosporins, are moving onto plasmids, and can easily mutate to significant activity against fourth generation cephalosporins suggest that AmpC xcex2-lactamases may very well constitute a potentially serious clinical threat.
Similar development of resistance can be seen in other antibiotic resistance genes such as the katG, rpoB, and rpsL genes of Mycobacterium tuberculosis which have resulted in multi drug resistance to isoniazid, rifampicin and streptomycin (C.D.C., xe2x80x9cOutbreak of Multidrug-resistant Tuberculosisxe2x80x94Texas, California, and Pennsylvania,xe2x80x9d MMWR 39:369-372 (1990); C.D.C., xe2x80x9cNosocomial Transmission of Multidrug-resistant Tuberculosis Among HIV-infected Personsxe2x80x94Florida and New York 1988-1991,xe2x80x9d MMWR 40:585-591 (1991); C.D.C., xe2x80x9cTransmission of Multidrug-resistant Tuberculosis from an HEV-positive Client in a Residential Substance Abuse Treatment Facilityxe2x80x94Michigan,xe2x80x9d MMWR 40:129-131 (1991)); adaptive virus resistance to antiviral agents as seen, for example, with HIV-encoded protease resistance to the protease inhibitors saquinavir, ritonavir, and indinavir (Condra et al., xe2x80x9cIn vivo Emergence of HIV-1 Variants Resistant to Multiple Protease Inhibitors,xe2x80x9d Nature 374:569-571 (1995)); antifungal resistance genes such as the atrc multiple drug resistance gene of Aspergillus nidulans (U.S. Pat. No. 6,060,264 to Skatrud et al.) and pdr1 multidrug resistance gene of Saccharomyces cerevisiae (Balzi et al., xe2x80x9cMe Multidrug Resistance Gene pdr1 from Saccharomyces cerevisiae,xe2x80x9d J. Biol. Chem. 262(35):16871-16879 (1987)); and the antimalarial resistance genes such as the pfmdrl gene of Plasmodium falciparum (Ruetz et al., xe2x80x9cThe pfmdrl Gene of Plasmodium falciparum Confers Cellular Resistance to Antimalarial Drugs in Yeast Cells,xe2x80x9d Proc. Natl. Acad. Sci. USA 93:9942-9947 (1996)).
One problem with conventional drug development strategies is that the pharmaceutical industry responds to a newly-emerged resistant strain by developing a modified version of the drug that was previously effective against the predecessor strain. Thus, the speed with which new drugs can be developed is limited by the fact that design of a new generation of a drug cannot begin until resistance has emerged and the properties of the resistant strains are known. In view of the limitations of a reactive strategy for conventional drug development, it would be more desirable to take a proactive approach for drug development. Hence, it would be very valuable to be able to predict the properties of resistant strains that will inevitably emerge and to begin designing the next generation of drugs in anticipation of that emergence.
The present invention is directed to overcoming these and other deficiencies in the relevant art.
One aspect of the present invention relates to a method of predicting the evolutionary potential of a mutant resistance gene. This method is carried out by providing a host cell which includes a mutant resistance gene either including two or more nucleic acid modifications or encoding a mutant polypeptide including two or more amino acid modifications, wherein the mutant resistance gene or mutant polypeptide confers a selectable advantage to the host cell, and then determining whether the mutant resistance gene is likely to evolve through two or more independent mutation events.
A further aspect of the present invention relates to a method of screening a drug for anti-pathogenic activity against a pathogen including a mutant anti-pathogenic resistance gene. This method is carried out by providing a host cell which includes a mutant anti-pathogenic resistance gene either including two or more nucleic acid modifications or encoding a mutant anti-pathogenic polypeptide which includes two or more amino acid modifications, wherein the mutant anti-pathogenic resistance gene or mutant anti-pathogenic polypeptide confers a selectable advantage to the host cell; growing the host cell on a selection media comprising a candidate drug or combinations thereof; and then determining whether the host cell is capable of growing on the selection media, wherein absence of host cell growth or proliferation indicates anti-pathogenic activity for the candidate drug or combinations thereof.
Yet another aspect of the present invention relates to a method of assessing the potential longevity of a candidate anti-pathogenic drug. This method is carried out by providing a resistance gene encoding a polypeptide which is ineffective against a candidate anti-pathogenic drug; introducing multiple mutations into the resistance gene to produce a mutant resistance gene which encodes a mutant polypeptide including two or more amino acid modifications, wherein the mutant polypeptide is effective against the candidate anti-pathogenic drug; and then determining the minimum number of mutations required to overcome the activity of the candidate anti-pathogenic drug, wherein the greater the minimum number of mutations, the greater the potential longevity of the candidate anti-pathogenic drug.
A still further aspect of the present invention relates to a mutant resistance gene prepared according to the process which is carried out by providing a resistance gene; introducing a plurality of mutations into the resistance gene to produce a mutant resistance gene which encodes a mutant polypeptide including at least two amino acid modifications, the mutant polypeptide conferring enhanced resistance to host cells including the mutant resistance gene; and then determining whether the mutant protein or polypeptide is likely to evolve through independent mutations.
Such mutant resistance genes include a coding sequence which includes a plurality of mutations, the mutant resistance gene encoding a mutant protein or polypeptide (i) including at least two amino acid modifications, (ii) conferring enhanced resistance to a host cell which expresses the mutant protein or polypeptide, and (iii) being likely to evolve through independent mutations.
Another aspect of the present invention relates to a mutant resistance-conferring protein or polypeptide derived from a resistance-conferring protein or polypeptide, the mutant resistance-conferring protein or polypeptide including at least two amino acid modifications relative to the resistance-conferring protein or polypeptide and being likely to evolve through independent mutations.
The present invention is directed primarily to the development of mutant resistance genes which exhibit increased resistance to conventional drug treatments. For example, with respect to an existing antibiotic resistance gene, such a gene is first mutated in accordance with the present invention to prepare a mutant antibiotic resistance gene that contains a plurality of mutations (i.e., either in the coding region or regulatory region) such that expression of the mutant antibiotic resistance gene confers to its host organism enhanced resistance against conventional antibiotic treatments. Once the mutant antibiotic resistance gene is obtained, it is then determined whether such a mutant antibiotic resistance gene is capable of evolving by a series of individual mutation events. This is determined by replicating discrete mutation events and then selecting for any enhanced activity (i.e., resistance) against conventional antibiotic treatments. This process is repeated by screening singly mutated antibiotic resistance genes, then doubly mutated antibiotic resistance genes, and so on, until it is determined whether the first obtained mutant antibiotic resistance gene is or is not likely to evolve under environmental selection. When it is determined that such a mutant antibiotic resistance gene or a multiply mutated antibiotic resistance gene is capable of evolving (i.e., in response to conventional antibiotics), then such a mutant resistant gene or multiply mutated resistance gene can be used to screen for next generation drugs. In this fashion, it is possible to identify next generation drugs before natural selection would allow for such drug development to occur. Moreover, it is possible to determine whether such next generation drugs are likely to provide long-lasting therapeutic treatment against organisms possessing mutant resistance genes. This determination can be made by assessing the number of discrete mutational events which would be required for an individual to overcome the effects of treatment by such a next generation drug. The greater the number of discrete mutational events which would be required to overcome the efficacy of such a drug, then the more likely that such a drug would have longand-lasting efficacy. This would enable drug manufacturers the opportunity to assess the potential profitability of one drug versus another.